Lecture 19

Ajouté : 2026-05-12

[00:00:03][music] [music] Hello everyone, welcome back to the MOO's course on advanced analytical technique. We have started unit second of this course and the topic that we are discussing is molecular luminous spectroscope.

[00:00:33]This is the last slide that we have ended our lecture with yesterday.

[00:00:39]And while discussing this slide, I was just explaining you in a nutshell about the Jablonsky diagram that we are having excitation through these blue lines and then if they are coming back directly from excited singlet to ground singlet it is fluesence.

[00:00:56]So while if they are under if the electrons are undergoing a flip but if they but if the electrons are undergoing a flip changing their spin state from excited singlet to excited triplet and then they are coming back to ground singlet state it is called as phosphorusence you all know about it and then this these phenomena and this phenomena can be represented in the form of a spectrum like this we have discussed this is the absorption spectrum which is due to which is due to the excitation. This is excitation spectrum which is due to the absorption this one while this is emission spectrum and the distance between these two maxima is called as the stroke shift.

[00:01:37]Let us start today's lecture with uh correlating the fluesence intensity with factors like the intensity of the incident light radiation as well as the concentration.

[00:01:53]So the intensity of the fluoresence emission depends upon various factors. For example, the intensity of the light that is being absorbed by the sample causing excitation.

[00:02:05]The same has been presented here that the radial power or the intensity of the fluoresence emission is proportional to the intensity of light that is being absorbed. And now how will we are going to calculate the light intensity of light being absorbed? Suppose P is the intensity of the incident light radiation. And if this P light of in intensity P not is passing through a sample while some of the energy is absorbed while the remaining is while the remaining light of intensity P is transmitted where P not where P is less than where P is of course less than P not because some of the intensity is being absorbed causing excitation of electron to the next level so that when electron comes they emit light radiation causing the phenomena of fluorescent Since some of the light is absorbed, the light that is being transmitted is having an intensity of P which is lesser than the incident radiations intensity P. Now in order to calculate the amount of light absorbed, we can use P minus P.

[00:03:09]So P minus P suppose gives us the light amount of light absorbed or the amount of power that is being transmitted into the electron by the absorption of that particular light radiation. So we can say that this fluoresence intensity is proportional to the difference in the light radiation of the incident light as well as the transmitted light because this difference gives us the amount of light that is being absorbed for excitation. P is the intensity of incident radiation while P is the intensity of transmitted light. Although we have nothing to do with transmitted light. I have explained you yesterday that even we do not want this transmitted light to reach to the detector and that is why in case of fluoresence spectroscopy we always keep the detector at 90° here we put we do not never put the detector here because if it is placed in the in a straight line it will always have some transmitted light causing current because of that transmitted light which is not fluoresence. So in order to calculate the fluoresence intensity only we always put this detector at 90° to the sample so that it will capture only the fluoresence emission because they are emitted in all directions. The intensity of this light also depend this is uh some calculation that I'm going to explain you just now the intensity of this fluoresence light also depends upon the concentration. So there are two parameters. One is the concentration on which the intensity of this fluorescent light depends. And then there is another parameter the difference in the intensity of the light radiation.

[00:04:47]Then there is one the fluence intensity also depends upon the concentration of the sample that is being under analysis.

[00:04:55]So there are two factors on which the fluence intensity depends. It depends upon the intensity of the light which is being absorbed. And this intensity is calculated by taking the a difference between the P and P. P not is the in intensity of incident radiation while P is the intensity of transmitted radiation. And it also depends upon the concentration of the species because if the concentration is high of course there will be more interaction there will be more molecules in the path way of the light radiation and there will when there is more interaction of course the absorption of light will be more. So when we remove the proportionality sign we put a constant K. So this is the equation. So this is the equation which relates the intensity of fluoresence with the intensity of light absorbed as well as the concentration of the sample.

[00:05:47]So C is the concentration of the sample and K is the proportionality constant.

[00:05:51]Now upon further solving this particular equation we have some more conceptual representation of the intensity of fluesence light. In one case we can have this intensity of fluoresence light is equal to ki epsylon c l and five where phi is the quantum yield which is defined as the ratio of intensity of fluoresence light or ratio of intensity of emitted light to the intensity of absorbed light.

[00:06:28]Also I explained you yesterday that there there's a term internal quantum efficiency which means that total number of electrons that are excited upon absorption of one light ray photon and then there is another term photoconducive gain. Photoconducive gain defines the increase in the current upon absorption of one single light photon.

[00:06:51]But the quantum yield in this particular case is defined as the ratio of the intensity of fluorescent light to the intensity of absorbed light. Now how this formula will now this equation has been evolved I hope that you reme I hope you remember about the beer Lambert's law. This is a very famous law. The beer lambert's law in UV spectroscopy. This law states that the decrease in intensity of the light radiation when it is passing through the sample with respect to thickness depends upon means it is proportional to the intensity of light radiation itself as well as concentration.

[00:07:53]If we rearrange this equation, we may have minus d upon upon dx is equal to k i and c. This is the same calculation that I I have made in this presentation.

[00:08:10]I'm showing you again.

[00:08:13]Now we rearrange this equation. Here we have removed the proportionality sign and we placed a constant k proportionality constant. Now if we rearrange this equation it is minus d i upon i is equal to minus k c dx.

[00:08:29]Upon integrating this equation, Upon integrating this equation we get so if we integrate this equation we get minus log I equals to minus K C and X and if we are integrating it within the limit I not to I so here we put I and here we put I not then this is will become log and there's a minus sign as well.

[00:09:10]log I upon log I minus it is natural log ln I not is equal to minus will go minus there's a minus sign as well so it minus k c and x it will become I upon I because log a minus log b is equal to log a upon b minus k c and x if we remove the log Here the other term will go to the power of E.

[00:09:47]So I will be equals to I E to the power minus K C X if intensity is represented by P in instead of I the equation will be P = P E power minus K CN X. Now since we have this equation and p is equal to p e ^ minus k cx putting this value of p from here this f will become k p minus p and this p is equal to p e to the power minus k cx and then we have this term F will be equal to if we take P common P common 1 - E ^ - K C X into into C.

[00:10:57]Now at low concentration, now at low concentration we get this form K P E C X and we put five also because it shows the quantum E. Quantum yield is basically always lesser than one which means that not all the light that is being absorbed is converted into fluorescent light. Some of the light is always been lost and that is why we get stoke shift. If the light is completely transformed into fluorescent light means the energy of the light that is being absorbed or the intensity of light that is being absorbed is 100% transformed into fluoresence. then the value of phi or quantum e will be one and it never and it never happens otherwise the peak position of absorption or excitation and emission will be same and stroke shifts will be zero and I told you that this is not the case with fluesence there is always a positive value of stroke shifts in case of fluesence so this is a more conceptual equation correlating the intensity of fluorescent light F with the intensity of incident radiation represented by I not or sometimes it is represented by P. And this equation also relates the intensity of fluoresence light with concentration C the quantum yield ph molar absorptivity as well as the power of the incident radiation P not or intensity of incident radiation I not both are same.

[00:12:48]So when we plot a graph of fluoresence intensity with analyte concentration for example we have a sample and we prepare different dilutions of this sample thereby changing its concentration.

[00:13:03]Suppose we have 1 ppm here then we have 2 ppm 3 ppm and like this just like in this case we have our solution is has been uh analyzed by fluoresence and the solution and those the solutions belongs to same sample only the concentration is different. So it is 1 mg per ml. 1 mg per ml is very high concentration because 1 mg per ml if we can multiply it with 1,000 it will be 1,000 mg per,000 ml which means 1,000 mg and 1,000 ml is 1 liter per liter which is equal to 1,000 ppm. So it is very high concentration.

[00:13:56]So it is so it is quite a high concentration.

[00:14:01]So if we plot a graph between the fluesence intensity as well as the concentration of the samples it will come like this. It will like come like this.

[00:14:14]In this graph you can observe that uh till this point or more correctly I should say till this point if we can see this line is straight while from this particular point the the graph is get getting deviated from the straight line and it is changing its position and finally it becomes almost constant.

[00:14:43]This shows that at low concentration, this shows that at low concentration, the fluoresence intensity F is proportional to C. There's a direct correlation.

[00:14:55]If we increase the concentration, fluence intensity will go up. But it happens up to a certain concentration limit. Beyond that concentration, this equation does not hold good. Here it is following linearity but then it becomes nonlinear.

[00:15:15]So at low concentration we have a linear relation between the fluesence intensity and concentration. As the concentration goes beyond a particular value the fluesence intensity does not increase correspondingly and at a certain point it becomes almost constant. Especially this particular region you can see it is getting constant and if we keeps on increasing the concentration to seven and 8 it will become constant.

[00:15:47]So this flow so this graph shows that so this plot shows that at low concentration we have a direct correlation and they follow the principle of linearity the fluence intensity and concentration but as the concentration goes up it deviates from the linearity it becomes nonlinear.

[00:16:05]Now the constant K that is being used in the equation on the previous page we have this equation and we also have a conceptual form where we have K I epsylon C L and five. So this proportionality constant K depends upon several factors. The first factor is the intensity of incident radiation itself. This constant K depends upon the intensity of fluoresence radiation.

[00:16:41]So K is proportional to P not. Now in order to enhance the intensity of fluoresence emission.

[00:16:51]What we should do?

[00:16:54]The first thing we should use a light radiation of high intensity. The intensity of the incident light should be high. The first thing because if the intensity of light radiation is high and since fluence intensity depends upon it directly it is directly proportional. It means that the chances of fluosense emission would be high.

[00:17:18]Further if the absorpt further if the absorptivity means this epsylon or sometimes this epsylon is represented by small a because we have another formula for absorption which is being used in UV spectroscopy absorbance is equal to a bc or sometimes it is written as epsylon bc where b is the path length C is concentration and this is molar absorptivity. Molar absorptivity is the absorption by one mole of the sample and it depends upon the sample. It is intrinsic property. It depends upon the type of sample. In case some samples have high mar absorptivity while some have low mar absorptivity. It depends basically it depends upon the presence of fluoro4 in the sample.

[00:18:15]So in order to increase the intensity of fluoresence radiation we must take a higher value of P not means the intensity of instant light should be high. Further the sample should have high molar absorptivity.

[00:18:27]It will increase the chances of fluoresence emission and the path length should and the path length should also be good enough. This L in this equation represents the path length. Here in this equation B represents the Here in this equation B represents the path length.

[00:18:45]Path length refers to the path length.

[00:18:49]If the cubid is placed like this or if the cubid is placed like this it is basically the thickness of the cubid or in this way it is the distance the light travels while passing through the sample. If this distance is long enough, the light radiation would have more time with samples to interact and there will be more number of molecules also that will come in on its way for interaction causing absorption.

[00:19:19]with high thickness. Then of course when light passes through this particular cuette there will be more number of molecules present along its path interaction would be more and ultimately the absorption would be more resulting into higher intensity of the fluoresence light.

[00:19:42]Again the quantum yield that that I have already explained you the quantum K uh the the constant K also depends upon the quantum efficiency. So this K depends upon intensity of the instant light maybe P 0 or it may be represented as I not clear this K also depends upon because K if K increases the chances of if because if K increases F increases. So if we take a sample with high molar absorptivity epsylon or a and if we take the thickness of the cub at high so b or the path length represent if it is represented by l should be high. These parameters are directly affecting the constant k. And then the last parameter that influences the constant is the quantum yield represented by this letter ph.

[00:20:37]The formula is given here. Quantum efficiency phi is equal to intensity of fluesence upon intensity of absorption.

[00:20:44]So it is a ratio of the intensity of fluoresence to the intensity of absorbance. And the value of phi is always lesser than when because in no case the intensity of light absorbed is 100% converted into the intensity of light emitted through fluoresence.

[00:21:01]Because when this light intensity is absorbed causing causing the sample to excite some of the light radiations are being lost in the form of non-radiative energy loss via heat or kinetic energy due to vibrational relaxations or due to quenching and the remaining energy only is emitted when the electron comes back.

[00:21:24]I have explained you these phenomena of quenching and vibrational relaxation when I was explaining the Jablonsky diagram in detail. There I have shown you these types of lines which basically represents the vibrational relaxations because in the ground electronic state also there are certain vibrational levels and in the excited electronic state also there are certain vibrational levels. So if the electron in So if the electron is so if the electron is traveling from ground state to excited state and it travels some vibrational levels as well.

[00:21:59]It comes from higher vibrational level to the lower vibrational level which is called as vibrational relaxation. It causes loss of energy in the form of heat and non a type of non-radiative energy loss. So this loss causes the intensity of the final fluoresence emission to be lower than the intensity of incident radiation and then sometimes quenching also occurs which is also a type of non-radiative energy loss. So these phenomena reduces the intensity of fluoresence light emission and therefore the value of phi is always lesser than one. These are the few factors that affect the intensity of the fluence light. quenching the temperature of the sample conjugation in the sample if present or not the pH of the system as well as the concentration of the samples. So these are the factors that we I'm going to discuss with you and that directly affects the intensity of the fluoresence light being emitted.

[00:22:59]uh as you all know that quenching refers to the loss of the intensity of light due to a non-radiative deactivation or non-radiative or non-radiative energy loss. And this happens either because this happens because of some quencher that is present in the sample that has been deliberately added or that is present that is present in the form of an impurity and this quencher reacts with the excited state. Excited state is always represented by a symbol star. So if A is in excited state showing fluesence and if there's a quencher present it will immediately becomes excited takes the energy from A while A gets deactivated and in this process since it is non-radiative no light is emitted and the intensity of fluence is reduced or sometimes it is completely vanished.

[00:23:52]So this is called as quenching. The quenching always reduces the intensity of the fluence emission. Then temperature. Temperature means for example if the temperature of the sample is high then that means then that means more number of molecules are colliding with each other because temperature is directly related to the kindinetic energy of the molecules. If temperature is low, the kindinetic energy is low which means that molecules are moving at slow speed and if at that speed the chances of collision are low and if the collision happens since the kindinetic energy low since the kindinetic energy is low the impact of those collisions is very low and therefore it will not cause any quenching or it will not cause any non-radiative loss. it will not affect the fluence intensity. But if the temperature of the system is high then of course there will be molecules which are moving at very fast speed and at that fast speed if they are colliding then it will create a significant impact transferring their their and transferring their energy thereby reducing the intensity and it causes the molecules to deactivate. It will causes a molecule which is excited to come back to the ground state. And in this case also no light is emitted only the energy is transferred from one molecule to the other molecule.

[00:25:21]Then conjugation molecule must have conjugation.

[00:25:25]Yesterday also I told you that presence of aromatic ring is a very good u thing for fluoresence to occur. Most of the fluorescent molecules are aromatic in nature. So if a sample is aromatic and if the conjugation is so if this so if the sample is aromatic of course it there are chances that it will show fluoresence and if in that aromatic ring the flloresence also depends upon the number of rings as well as the conjugation. So if we have more conjugation the electrons due to this conjugation are deoized. They can these electrons can move freely and these freely moving electrons can be excited easily to the excited state and when they come back they emit light radiation in the form of fluorescent light. So conjugation is directly proportional to the fluescent intensity. we have conjugation in the system. It will of it will of course increase the intensity of the fluorescent light and because and this conjugation causes resonance structure and resonance stabilizes the excited state. For example, in case of eneline, this is a molecule of enoline and we have a lone pair of electron on nitrogen. When this lone pair is involved in the process of fluoresence like this, it forms a double bond and then the lone pair will come over here and we have a double bond and in this way these electrons are deoized means the excited state and deoization on resonance means the molecules loses some energy due to this resonance structure and therefore it is stabilized. Now since the molecule is stabilized the electrons can be easily excited by absorption of light energy and when they come back they emit light radiations of longer wavelength means the visible light and thus they show the phenomena of fluoresence. So the molecule must have conjugation and as the conjugation increases the fluence intensity increases because conjugation causes electrons to flow freely and those electrons can be very easily excited by absorption of energy and this conjugation stabilizes the molecule because of the phenomena of resonance.

[00:27:40]Then comes the pH of the system. pH has some complicated effect on the phenomena of fluesence. uh but in general we say that pH increase causes the fluoresence intensity to go up. Clear? And in case of acidic samples or samples with lower pH that there has been some quenching the intensity of the fluorescent light decreases. Why? Because for example if you have a sample which has a lone pair of electron just in case of for example we have this particular case we have a lone pair of electron if it is present in an acid acid will donate its proton it will form this species because under acidic condition the proton is being released by an acid and which is taken by the non-bonded electron pair. For example, in this particular case, since any has a lone pair of electron and if the solution is acidic, it will release proton. That proton approaches to the non-bonded electron pair making it like this. Converting this molecule into this particular form. Now the electron pair is not free to move giving the phenomena of resonance and since it is not involved in the phenomena of resonance.

[00:29:11]It is not moving at all. It is it is stays it is stick to its place. Then that means there will be no fluoresence in this particular case. So uh low pH value means an acidic solution reduces the intensity of fluoresence light. Although in some cases even in basic solutions also we have a lower fluoresence intensity. So the effect of pH is a little bit complicated but as a in general we can say that up to a certain limit the in acidic conditions we have a lower intensity of fluence and as the pH goes up means we are moving towards basic solutions the intensity goes up.

[00:29:56]The same has been represented in this diagram. Here you can see that we are changing the pH of a solution and the fluoresence intensity although it decreases initially but in all cases the fluoresence intensity is increasing as we are moving from lower pH to higher pH. As we are moving from acidic solution to alkaline solution the intensity of the fluoresence is going up. In this particular case, we have used 2 mm concentration and the excitation of this surfactant means the absorption of light happens at 515 nanometer. While the emission because emission always occurs at longer wavelength, it is always towards the right hand side. So emission occurs at 570 nanometers. So there is a difference in the intensity of 55 nanometers. The stroke shift is 55 nanometers.

[00:30:51]And in this particular case of surfectant of 2 mm concentration the effect of pH has been represented as the acidic solutions have lower fluorosense intensity. While as we move towards higher pH from lower pH value as we are means we are moving towards basic solutions the intensity of the fluence is going up.

[00:31:14]This is again the same formula that I have divi derived in the previous case.

[00:31:20]the effect of concentration and it shows that fluesence is directly proportional to concentration but since I have shown you a graph as well this equation holds good at lower concentration only because if we increase the concentration at high concentration the graph moves away from linearity it becomes nonlinear while at low concentration it is linear means fluoresence intensity is directly proportional to concentration. So we can say that just like pH we have a little bit complicated effect of concentration on fluence intensity at lower concentrations means for dilute solution the fluence is directly proportional to the concentration we have a linear relation as we keeps on increasing concentration there will be a corresponding increase in the fluence intensity but after a particular value of concentration there has been some deviation from this linearity and at very high concentration This plot becomes almost parallel to x-axis. Means when the concentration becomes very high there will be no increase in intensity and sometimes due to high concentration there will be some sort of quenching and the intensity of fluosence decreases at very high concentration.

[00:32:41]The same can be represented in this graph. Here you can see that at lower concentration the line is straight which means that up to 200 mg or 300 mg per liter means up to 300 ppm concentration of the sample we are having a linear relation where fluoresence is proportional to C its concentration. But as the concentration goes up to 400 ppm 600 and 800 the graph is moving away from linearity and the and the intensity is not correspondingly increase with increasing concentration and at 800 it becomes almost constant and I and as I said that at very high concentration the molecules collide with each other the number of collisions increases causing the number of collisions increases and thereby the intensity of fluence decreases due to quenching. And the same is represented here that at 1,000 ppm means 1 g per liter 1,000 mg per liter the concentration is so high that in in this particular case that because it it is the fluoresence of crude oil. So at very high concentration of crude oil there are collision between the molecules and therefore the concent fluence intensity and therefore the fluence intensity decreases means self quenching is taking place.

[00:34:03]So this is all about the effect of different factors on fluence intensity.

[00:34:09]In the next slide we'll discuss some more details of the phenomena of fluesence. Till then thank you very much. [music]